Interaction Between Mast Cells and Perineurial Fibroblasts in Neurofibroma

Path. Res. Pract. 183, 453-458 (1988)

Interaction Between Mast Cells and Perineurial Fibroblasts in Neurofibroma New Insights into Mast Cell Function C. J. Kirkpatrick and A. Curry Department of Pathology, University of Manchester and Public Health Laboratory, University Hospital of South Manchester, England

SUMMARY Transmission electron microscopy was performed on seven neurofibromata to study the relationship between the frequently occurring mast cells and the other cell types present in the tumour. Intimate association was observed between mast cells and perineurial fibroblasts, but not between mast cells and Schwann cells. At the contact interface between mast cells and perineurial fibroblasts, numerous vesicle-like structures were observed with corresponding endocytotic vesicles in the fibroblast plasma membrane. The authors regard this close morphological relationship as in vivo evidence for a possible role ofmast cells in fibroblast metabolism, a conclusion which has already been drawn from in vitro studies, but until now inadequately supported by observations in vivo.

Introduction The presence of considerable numbers of mast cells in neurofibromata is common knowledge amongst histopathologists and was documented as early as 1911 by Greggio12. In an extensive review on mast cells in the nervous system, 0lssoo 17 discussed possible roles for these cells in demyelination and the activation of phagocytes in injured nerves. On the other hand, Gamble and Goldby10 suggested that mast cells may be involved in the metabolism of endoneurial connective tissue. The latter hypothesis has been greatly strengthened by analytical studies on mast cells, demonstrating that they contain substances, e.g. r.roteases, capable of altering connective tissue integrity' 15. Furthermore, investigations in vitro indicate that fibroblasts are able to phagocytose mast cell granules 20 and that very close contact between isolated mast cells and cultured fibroblasts can result in "transgranulation", transfer of granules from the former to the latter cell type ll . Most of our knowledge on mast cell biology is derived, as in the latter two studies, from investigations on rodent mast cells. In the ultrastructural work presented here we have used neurofibromata as a rich source of mast cells from a human pathological situation to demonstrate that © 1988 by Gustav Fischer Verlag, Stuttgart

there is evidence for intimate association between mast cells and perineurial fibroblasts in vivo. In addition, ultrastructural evidence will be presented to show that this intimate relationship may be accompanied by metabolic interaction. Material and Methods Seven neurofibromata were studied ultrastructurally and were taken from subcutaneous locations in patients with either the single lesion or multiple tumours as part of von Recklinghausen's neurofibromatosis. Specimens were fixed in Carson's buffered formalin, which gives excellent preservation of morphology at both light and electron microscopicallevels4• After routine processing to paraffin wax, sections were cut at 4 Itm and stained with haematoxylin and eosin for light microscopy. Material for conventional transmission electron microscopy was post fixed in 1% (w/v) osmium tetroxide, dehydrated in a graded series of alcohols followed by propylene oxide and embedded in Agar 100 resin (Agar Aids). 1ltm sections were cut from the resultant blocks, mounted on glass microscope slides and stained with toluidine blue. Ultrathin sections were cut from suitable areas, mounted on uncoated 200 mesh copper grids and stained with uranyl acetate followed by lead citrate. Grids were examined in an AEI EM 801 electron microscope. 0344-0338/88/0183-0453$3.50/0

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Results

Light Microscopy

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All seven neurofibromata were classified as Type II according to Weiser21 and showed a poorly circumscribed tumour consisting of spindle-shaped cells with frequent "comma"-shaped nuclei set in a collagenous stroma. Even with haematoxylin and eosin the numerous mast cells were apparent in the stroma, both close to and away from the frequent small capillaries.

Transmission Electron Microscopy At ultrastructural level three principal cell types were seen in a dense collagenous matrix with frequent capillaries, namely Schwann cells with their characteristic continuous basal lamina, numerous fibroblast-like cells and mast cells (Fig. 1). These fibroblast-like cells, or perineurial fibroblasts according to Erlandson8, were characterised by a large nucleus and long, thin cytoplasmic processes which penetrate the collagen matrix, but do not possess a basal lamina (Fig. 2). Whilst an occasional mast cell was situated in the collagenous matrix with no apparent

Fig. 2. Perineurial fibroblast (F) with long cytoplasmic processes surrounded by collagenous matrix and numerous Schwann cells (SC). x 5250.

Fig. 1. Low power view of neurofibroma collagenous matrix containing a Schwann cell (SC) with surrounding basal lamina (arrowheads), a fibroblast-like cell (F) and a mast cell (MC). Note the fibroblast cytoplasmic process partially enveloping the mast cell. x 8 400.

contact with other cell types, the majority showed intimate association with perineurial fibroblasts, but never with Schwann cells (Fig. 3 a). Mast cells gave the classical ultrastructural picture of a centrally placed nucleus with peripherally condensed chromatin, and numerous granules with a poorly defined limiting membrane, which show the various characteristic morphological forms, namely "scrolls", "fingerprints" and electron-dense, patchy "crystalloid" structures (Fig. 4). The plasma membranes of the mast cells demonstrate numerous short cytoplasmic extensions or lamellopodia 22 • These lamellopodia can increase in length and interdigitate with the long cytoplasmic extensions of perineurial fibroblasts which frequently partially invest the mast cells (Figs. 1,3 a and 5). The most striking feature of the intercellular spaces between these closely interacting cells was the presence of numerous vesicle-like structures (mean diameter approximately 75 nm) (Figs. 3 b, 6). Fig. 7 shows that these structures were most apparent along those parts of the mast cell periphery in apposition to the cytoplasmic processes of the perineurial fibroblasts. Fig. 6 provides structural evidence that some of these vesicle-like structures may be arising by budding from the

Interaction Between Mast Cells and Perineurial Fibroblasts in Neurofibroma . 455

mast cell plasma membrane by a process of exocytosis. These structures also come into direct contact with the adjacent fibroblast plasma membrane (Fig. 6). In addition the apposing plasma membrane of the perineurial fibroblasts shows the formation of endocytotic vesicles. It is important in this context to stress that on no occasion was there total degranulation of a mast cell or even the exocytotic release of an intact mast cell granule. Despite the close proximity of these two cell types, only one junctional complex was observed between a mast cell and a perineurial fibroblast (Fig. 8). Discussion In this paper we have shown that transmission electron microscopy on neurofibromata reveals intimate association between mast cells and perineurial fibroblasts, but not between mast cells and the other major cellular component of neurofibromata, the Schwann cells. At this point it is important to briefly mention the controversy surrounding the exact nature of what is termed "perineurial fibroblast" in this paper. Whilst Lever and Schaumburg-Lever16 regard perineurial cells as present in significant numbers only at the site at which the cutaneous nerve enters the neurofibroma,

Erlandson and Woodru££9 claimed that the predominant cell type in neurofibromata had perineurial cell characteristics, such as long, thin, bi- and tripolar cell processes with fragmented or partially absent external lamina, short profiles of rough endoplasmic reticulum, ribosomes, prominent microfilaments and pinocytotic vesicles. In our study, the fibroblast-like cells in the neurofibromata did not possess even a fragmented external (or basal) lamina. Erlandson and Woodruf£9 regarded classical fibroblasts as rare in neurofibromata and suggested that the "fibroblasts" described by Lassmann et al. 14 in plexiform neurofibromata represented intermediate forms between perineurial cells and fibroblasts. The in vitro evidence that Schwann cells can synthesize procollagen2,5 has led Erlandson and Woodruf£9 to deem it unnecessary to invoke the presence of fibroblasts to account for collagen production in neurofibromata. Whatever the origin of the fibroblast-like cellular component of neurofibromata may be, our study shows that it is this cell, and not the mature Schwann cell with its continuous well-developed basal lamina that forms a close association with the mast cell. In 1965 Pineda 19 described the ultrastructural characteristics of mast cells in neurofibromata, but did not describe any association with the other cellular elements present. Heine and Forster 13 described an intimate rela-

Fig. 3. a. Two perineurial fibroblasts investing a mast cell, x 3000. b. High power view of the intimate association between the mast cell and the cytoplasmic process of the fibroblast. Note the presence of numerous vesicle-like structures in the intercellular space (arrowhead). x 7600.

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c. J. Kirkpatrick and A. Curry In this case the degenerating mast cell was reported as showing free granules adjacent to areas of loosening of collagen. The latter was interpreted as evidence of fibrolysis. Degranulating mast cells were also a prominent feature in ileal specimens of Crohn's disease studied by Dvorak et aU who detected mast cells adjacent to and within proliferating autonomic nervous system components. One important difference between these studies and our investigations is that we did not observe release of intact granules from mast cells. Instead, the interface between the mast cell and the interacting perineurial fibroblast consisted of complex plasma membrane interdigitations between which vesicle-like structures, much smaller than intact mast cell granules, were seen. If, in fact, these structures are true vesicles and not merely cross-sections of closely-packed plasma membrane lamellopodia, then they could indicate that mast cell granule content is transferred by a process of exocytosis to the neighbouring fibroblast. Some evidence has been found for a budding process from the surface of a mast cell (Fig. 6). Furthermore, the reciprocal change seen in the interacting fibroblast, namely endocytosis, is strong morphological evidence that this intimate interaction is more than mere topographical association, but may truly represent a metabolic interaction.

Fig. 4. High power view of mast cell showing typical mast cell granules, containing electron-dense crystalloid structures (arrows) and scroll-like contents. In addition, interdigitation of mast cell processes with fibroblast processes is seen. Note budding of vesicle-like structures from the mast cell surface (arrowhead). X 45000.

tionship seen at light microscopical level between mast cells and unmyelinated preterminal nerve fibres in human subcutis and canine myocardium. More recently, Wiesner-Menzel et al. 22 reported a very close morphological relationship between mast cells and non-myelinated nerve fibres in a subungual glomus tumour. These mast cells showed lamellopodia or broader cytoplasmic extensions which were directly apposed to axons. In addition, these authors stated that there was evidence of exocytosis of granules, although this statement was not adequately documented by electron photomicrographs. Our ultrastructural observations indicate that exocytosis might indeed be a feature of any mast cell which adopts close cellular association with another cell type, in this case with the perineurial fibroblast of the neurofibroma. However, the vesicle-like structures which we have described might equally represent cross-sections of the lamellopodia which are characteristically present on mast cell surfaces 22 • Close association between mast cells and fibroblasts has also been described in a juvenile nasopharyngeal fibroma 1.

Fig. 5. Low power view of mast cell interdigitating with cytoplasmic process from a perineurial fibroblast. x 23000.

Interaction Between Mast Cells and Perineurial Fibroblasts in Neurofibroma . 457

Concerning this latter point, in vitro studies provide evidence to support the hypothesis that mast cells can alter connective tissue metabolism. Thus, Greenberg and Burnstock ll used time-lapse cinephotomicrography and transmission electron microscopy to show that in the rat, isolated mast cells can interact with cultured fibroblasts and endothelial cells with resulting transfer of mast cell granules to the recipient cell and endocytosis in the latter cell. Subba Rao et aJ.2° demonstrated the ability of cultured rat embryonic skin fibroblasts to phagocytose rat mast cell granules either added to the culture medium or released from the intact mast cell using anti-rat IgE or compound 48/80. This phagocytotic process was also shown to be followed by increased secretion of collagenase into the medium.

Fig. 6

Fig. 8. A junctional complex (arrowhead) seen between a mast cell and a closely apposed perineurial fibroblast. x 55000. Fig. 6. Intercellular space between a mast cell and a cytoplasmic process from a perineurial fibroblast. This space is occupied by numerous vesicle-like structures which may represent cross-sections of lamellopodia from the mast cell surface. Note the pinocytotic vesicles within the fibroblast cell process (arrowheads). x 32400.

Fig. 7

Fig. 7. Wedge-shaped profile of a mast cell situated between two cytoplasmic processes of perineurial fibroblasts. Note presence of vesicle-like structures (arrowheads) between these interacting cells, but absence of them from the surface facing the Schwann cell (sq. x 8000.

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In conclusion, it is worthy of mention that the best known reaction of mast cells in disease, namely massive degranulation in response to IgE-antigen complex formation, probably represents only one end of a spectrum of metabolic alterations by the mast cell. This has been well argued by Padawer l8 who presented a. number of experimental observations leading to the conclusion that "mastcell degranulation and release of amines are not normal events, and that they are in fact antithetical to the proper functioning of the mast cell". Our study presents morphological findings in a pathological state which demonstrates intimate mast cell contact with fibroblasts not involving mast cell degranulation, but with apparent reciprocal endocytosis by the intimately associated fibroblastic cells. It is also interesting that the extensive plasma membrane fold formation in the mast cells which interact with the fibroblasts had been observed by Burwen and Satir3 in isolated rat peritoneal mast cells undergoing exocytosis. They postulated that the process of exocytosis provided the source of membrane required for fold formation. Our observations represent an in vivo correlate of these in vitro functional studies and provide morphological evidence that in the neurofibroma at least, a close association exists between mast cells and fibroblast-like cells. Acknowledgements The authors would like to express their gratitude to Mrs. Sandra Applegate and Miss Patricia Rowland for their excellent technical assistance and to Mrs. Anne Mellor and Mrs. Petra Fischer for kindly typing the manuscript.

References 1 Arnold W, Huth F (1978) Electron microscopic findings in four cases of nasopharyngeal fibroma. Virchows Arch B Cell Pathol379: 285-298 2 Bartlett Bunge M, Williams AK, Wood PM, Uitto 1, 1effrey 11 (1980) Comparison of nerve cell and nerve cell plus Schwann cell cultures with particular emphasis on basal lamina and collagen formation. 1 Cell Bioi 84: 184-202 3 Burwen S1, Satir BH (1977) Plasma membrane folds on the mast cell surface and their relationship to secretory activity. 1 Cell Bioi 74: 690-697 4 Carson FL, MartinJH, LynnJA (1973) Formalin fixation for electron microscopy: Are-evaluation. Amer J Clin Path 59: 365-373

5 Church RL, Tanzer ML, Pfeiffer SE (1973) Collagen and procollagen production by a clonal line of Schwann cells. Proc. Natl Acad Sci USA 70: 1943-1946 6 Dabbous MKH, Walker R, Haney L, Carter LM, Nicolson GL, Woolley DE (1986) Mast cells and matrix degradation at sites of tumour invasion in rat mammary adenocarcinoma. Br J Cancer 54: 459-465 7 Dvorak AM, Monahan RA, Osage JE, Dickersin GR (1978) Mast cell degranulation in Crohn's disease (Letter to Editor) Lancet 1: 498 8 Erlandson RA (1985) Peripheral nerve sheath tumours. Ultrastruct Pathol 9: 113-122 9 Erlandson RA, Woodruff JM (1982) Peripheral nerve sheath tumors. An electron microscopic study of 43 cases. Cancer 49: 273-287 10 Gamble HJ, Goldby S (1961) Mast cells in peripheral nerve trunks. Nature (Lond) 189: 766-767 11 Greenberg G, Burnstock G (1983) A novel cell-to-cell interaction between mast cells and other cell types. Exp Cell Res 147: 1-13 12 Greggio H (1911) Les cellules granuleuses (Mastzellen) dans les tissues normaux et dans certaines maladies churgicales. Arch Med Exp 23: 323 13 Heine H, Forster FJ (1975) Relationships between mast cells and preterminal nerve fibres. Z Mikrosk Anat Forsch 89: 934-937 14 Lassmann H, Jurecka W, Lassmann G, Gebhart W, Matras H, Watzek G (1977) Different types of benign nerve sheath tumors. Light microscopy, electron microscopy and autoradiography. Virchows Arch A Pathol Anat 375: 197-210 1 Lee TDG, Swieter M, Bienenstock 1, Befus AD (1985) Heterogeneity in mast cell populations. Clin Immunol Rev 4: 143-199 16 Lever WF, Schaumburg-Lever G (1983) Tumors of neural tissue. In: Histopathology of the Skin, 6th Ed. JB Lippincott Co., Philadelphia, pp 677-680 17 Olsson Y (1968) Mast cells in the nervous system. Int Rev Cytol 24: 27-70 18 Padawer J (1979) Mast cell structure: implications for normal physiology and degranulation. In: Pepys J, Edwards AM (eds) The Mast Cell in Health and Disease. Pitman Medical, London, pp 1-8 19 Pineda A (1965) Mast cells - their presence and ultrastructural characteristics in peripheral nerve tumours. Arch Neurol 13: 372-382 20 Subba Rao PV, Friedman MM, Atkins FM (1983) Phagocytosis of mast cell granules by cultured fibroblasts. 1 Immunol 130: 341-349 21 Weiser G (1978) Neurofibrom und Perineuralzelle. Electronenoptische Untersuchung an 9 Neurofibromen. Virchows Arch A Pathol Anat 379: 73-83 22 Wiesner-Menzel L, Schulz B, Vakilzadeh F, Czarnetzki BM (1981) Electron microscopical evidence for a direct contact between nerve fibres and mast cells. Acta dermatovener 61: 465-469

Received August 14, 1987· Accepted in revised form January 18, 1988

Key Words: Mast cells - Neurofibroma - Fibroblast - Perineural cells - Cell-cell interaction Prof. C.

J. Kirkpatrick, Abteilung Pathologie, Klinikum der RWTH, PauwelsstraBe, D-5100 Aachen, FR Germany